Fluorescence fluctuation spectroscopy (FFS) is an attractive technique for cellular applications. It determines kinetic and molecular properties of proteins with submicron resolution and single molecule sensitivity. A unique feature of FFS is the ability to measure the stoichiometry and binding curve of fluorescently labeled protein complexes through brightness analysis. Brightness is the average fluorescence intensity of a single protein complex and is directly proportional to the number of labeled protein molecules. Soluble homo- and hetero- protein complexes have been successfully characterized by brightness analysis of cellular FFS data. While we have made enormous progress in the characterization of soluble protein complexes by FFS, our ability to investigate membrane-bound proteins by FFS remains woefully inadequate. This deficit is especially egregious because more than half of all proteins interact with the membrane. Data from structural studies show that membrane proteins function in complexes, but our ability to detect and quantify the interactions is very limited. This proposal seeks to extend FFS capabilities to the characterization of membrane-bound proteins by capitalizing on recent advances in FFS methodology. We first focus on proteins at the plasma membrane. The technical approach is based on z-scan FFS where the optical observation volume is scanned axially through the sample. Z-scan FFS takes the geometry of the sample into account and separates between cytoplasmic and membrane signal. We develop and characterize the performance of z-scan FFS and extend the technique to include correlation functions, lateral imaging, and fluorescence lifetime. Both single- and dual-color z-scan FFS are developed in order to characterize both homo- and hetero-protein interactions. In addition we will explore the potential of FFS to characterize proteins at vesicles inside the living cell. Vesicles transport, sort, digest and stor proteins. The regulation of these diverse processes is not well understood but involves specific proteins that associate with vesicles. FFS experiments of such vesicles carrying fluorescently-labeled proteins lead to bright, but infrequent peaks on top of background. Characterization of such data is a daunting challenge, but recent advances in brightness analysis offer a quantitative approach to separate the background from the bright peaks. We will investigate this approach with the goal of determining the copy number of proteins and the coexistence of two proteins on the same vesicles. This development of new FFS methods fills a critical need, because we still lack methods that quantify proteins at membranes and at vesicles. The impact of the new methodology will be felt in many biological areas with applications ranging from basic research in cell biology to pharmaceutical drug screening. In vivo FFS could help fighting diseases by providing detailed information about protein interactions and may lead to the identification of targets for drug development. PUBLIC HEALTH RELEVANCE: The goal of the project is the development of a spectroscopic method with the unique ability to quantify protein interactions inside living cells. Knowledge of proteins and their interactions is a prerequisite for the identification of the molecular mechanism underlying a disease and provides crucial information for rational drug design.